Antibody therapeutic agents show less side effects and high treatment efficacy together in the treatment of various diseases compared to chemical agents, and thus global pharmaceutical companies and bioengineering companies intensively invest on the development thereof. At present, a large number of antibody therapeutic agents are used in clinical applications, and many therapeutic agent candidates are under clinical trials.
However, despite such advantages, the antibody therapeutic agents have problems in that production cost is high, it is difficult to escape the existing patent barriers, it is difficult to penetrate into cells due to their large molecular weight, and therapeutic effects of patients are not actually high as expected. Accordingly, in recent years, the development of therapeutic agents based on small-sized non-antibody protein scaffold for replacing the antibody therapeutic agents has been actively conducted. Many studies have revealed that such small-sized non-antibody protein scaffolds have an advantage over the antibody therapeutic agents in that they penetrate cancer tissue with greatly increased efficiency, suggesting that they can offer improved therapeutic effects.
Under this background, the present inventors successfully developed a repebody which is a non-antibody protein scaffold capable of replacing existing antibodies. It was shown that the repebody has a size equal to about ⅕ of that of the immunoglobulin antibody, is produced in large amounts in E. coli, and shows little or no immunogenicity as a result of animal tests (Korean Patent No. 1,517,196). In addition, it has been demonstrated that the repebody has very high thermal and pH stabilities, and is able to very easily increase its ability to bind to a target molecule up to a picomole level, and has remarkably superior specificity to the target molecule is very high (Korean Patent No. 1,356,075, Korean Patent Laid-Open Publication No. 10-2013-0007380).
Vascular endothelial growth factor (VEGF) is a disease-inducing factor which is overexpressed in various cancer cells, including metastatic colorectal cancer cells, to induce angiogenesis, thereby promoting tumor growth and metastasis. It was reported that VEGF is overexpressed in not only tumors, but also blood vessel-related diseases such as age-related macular degeneration (AMD). As such, VEGF overexpression is related to various diseases, and thus studies on the development of therapeutic agents targeting VEGF have been actively conducted.
At present, protein-based drugs targeting VEGF for treatment of age-related macular degeneration (AMD) include Bevacizumab that is a monoclonal antibody, but it was reported that Bevacizumab has lower binding affinity and therapeutic efficacy than other new monoclonal antibody drugs. To overcome this disadvantage, the development of new therapeutic methods, including use in combination with chemotherapy and the development of new drugs, has been attempted. Other VEGF-targeting drugs include Ranibizumab which is a monoclonal antibody fragment (Fab) derived from Bevacizumab. Ranibizumab has been used as an agent for treating age-related macular degeneration, but was reported to have a side effect and limited therapeutic efficacy. Recently, Aflibercept, which is a recombinant protein composed of the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of the human IgG1 immunoglobulin, has been developed and clinically used. Aflibercept is known to offer improved efficacy toward AMD compared to other protein-based drugs, but has some side effects and very expensive. Therefore, there is an urgent need to develop therapeutic agents targeting VEGF for treatment of AMD with a new concept, which overcome the limitations of existing drugs.
The present inventors have successfully developed a specific protein binder for various disease-related target proteins including VEGF by use of the above-described repebody scaffold, and have verified through a cell-based method that the specific protein binder has biological inhibitory effects (Korean Patent No. 10-1517960). However, studies on the application of the specific protein binder are still in the beginning stage, and thus further studies are actively underway.
Under this background, the present inventors have made extensive efforts to successfully obtain a protein that binds specifically to VEGF, which is an important target protein for treatment of age-related macular degeneration and various cancers, using the above-described repebody scaffold, and as a result, have screened a novel polypeptide having a specific binding affinity for VEGF, based on a random mutation library constructed based on the analysis of the structural characteristic (modularity) and overall structure of the repebody. In addition, the present inventors have screened and produced a polypeptide, which is a novel repebody that binds specifically to VEGF, through a beneficial mutation predicted based on a protein complex structure, and have found that the repebody is capable of binding specifically to VEGF and offers therapeutic efficacy in the choroidal neovascularization (CNV) animal model, thereby completing the present invention.